1. Field of the Invention
This invention relates to a delivery system that releases a diagnostic marker or therapeutic agent in a manner sensitive to the concentration of urease in a physiological compartment, particularly the gastrointestinal tract.
2. Background Information
Helicobacter pylori (previously known as Campylobacter pylori) has been shown to be etiologically associated with gastritis, duodenitis, gastric and duodenal ulcers (Dooley, CP. Background and historical considerations of Helicobacter pylori. Gastroenterol. Clin. North Am. 1993;22:1-4). Patients with these conditions may not be cured unless the organism is eliminated, usually by the use of a combination of antibiotics (Tytgat et al. Helicobacter pylori infection ad duodenal ulcer disease, Gastroenterol. Clin. North Am., 1993;22:127-140). Therefore, there is a need for a simple and reliable marker for Helicobacter pylori infection so that it can be easily diagnosed and the response to therapy be readily evaluated.
At present the most commonly used method for detecting the presence of Helicobacter pylori is by placing a flexible tube with a light into the patient's stomach (a procedure called endoscopy) and obtaining biopsy samples of the stomach. The samples are then cultured as well as examined histologically to look for Helicobacter pylori. This method is expensive, and in addition, places the patient at risk for complications from the endoscopy (Chodos et al. Campylobacter pylori and Gastroduodenal Disease: A Prospective Endoscopic Study and Comparison of Diagnostic tests. Am. J. Gastroenterol. 988;83:1226-1230). Noninvasive tests therefore have been developed to overcome some of these problems. One of these is a serological antibody test which measures the blood level of IgG antibody to Helicobacter pylori. The antibody level becomes elevated in response in Helicobacter pylori infection. However, a major limitation of this test is that antibody may persist in the blood even after elimination of the organism. This test is therefore not useful for determining response to therapy and cannot distinguish active infection from infection in the remote past (Brown et al, Diagnosis of Helicobacter Pylori Infection. Gastroenterology Clinics of N. Am. 1993;22:105-115).
U.S. Pat. No. 4,830,010 (Marshall) discusses another noninvasive test for the diagnosis of Helicobacter pylori that relies on the measurement of carbon dioxide (CO.sub.2) in the breath. This test is based on the fact that the bacteria possesses the enzyme called urease, which breaks down urea into CO.sub.2 and ammonia. To perform this test, urea, labeled with either .sup.13 C or .sup.14 C, is orally ingested by the patient. Subsequently, breath samples are collected at 30 and 60 minutes. The orally ingested urea is broken down to CO.sub.2 and ammonia by the bacteria in the stomach. CO.sub.2 is rapidly absorbed into the blood and then excreted via the breath. The method used for measurement of this CO.sub.2 in the breath depends on the initial labeling of the urea. If .sup.13 C-urea (nonradioactive) is used, .sup.13 CO.sub.2 concentration in the expired breath is measured by gas isotope spectrometry (Brown et al, Diagnosis of Helicobacter Pylori Infection. Gastroenterology Clinics of N. Am. 1993;22:105-115). Use of .sup.14 C-urea results in the production of radioactive .sup.14 CO.sub.2 which is measured in the breath using a scintillation counter. Both methods share a common disadvantage in that they require specially trained individuals to administer the test and special breath collection devices, and patients may have to follow potentially unwieldy instructions.
In addition to the general drawbacks associated with breath tests, each of the two breath tests has its own unique problems. The use of .sup.14 C-urea requires that radioactive material be administered to the patient. The material is expensive and a scintillation counter is required for analysis (Brown et al., Diagnosis of Helicobacter Pylori Infection. Gastroenterology Clinics of N.Am 1993;22:105-115; Graham, What you should know about the methods, problems, interpretations, and uses of urea breath tests. Am. J. Gastroenterol. 1991;86:1118-1122).
The use .sup.13 C-urea has the disadvantage that the detection of .sup.13 CO.sub.2 requires a mass spectrometer, an instrument that is expensive and is not readily available in all parts of the world.
A variation on the above theme involves the detection of .sup.13 C-bicarbonate in the serum after ingestion of .sup.13 C-labeled urea (Moulton-Barret et al. Serum .sup.13 C-Bicarbonate in the assessment of gastric Helicobacter pylori urease activity. Am. J. Gastroenterol. 1993;88:369-74). This method is based on the fact that the .sup.13 CO.sub.2 produced by the action of urease on .sup.13 C-labeled urea is present in the blood in the form of bicarbonate formed as a result of a reversible reaction with water: EQU CO.sub.2 +H.sub.2 O=H.sup.+ +HCO.sup.3-
However, this method is technically demanding and, in addition, bicarbonate levels in the serum may fluctuate in response to many variables such as the acid-base balance in the blood.
The known ability of Helicobacter pylori to split urea is the basis for yet another test, one which requires the use of biopsy samples obtained at endoscopy. For this test, a biopsy is obtained from the patient's stomach and the specimen tested for the presence of urease (Thillainayagam et al: A Novel Enzyme Radioimmunoassay for Serodiagnosis of Helicobacter Pylori Infection. Gut 1991;32:467-469). Although this method gives rapid results, the requirement for endoscopy to obtain the biopsy samples is a major drawback.
Most of the above tests for Helicobacter pylori have mainly concentrated on the production of CO.sub.2 by the action of urease on ingested urea. In contrast, there are fewer reports on the detection of ammonia which is generated along with CO.sub.2 in the same reaction (Urea-.fwdarw.CO.sub.2 +2NH.sub.3). Marshall has patented a method for detection of Helicobacter pylori by the measurement of either CO.sub.2 or ammonia in the breath after a urea meal (U.S. Pat. No. 4,830,010). Other workers have shown that urine testing for .sup.15 N-ammonia after ingestion of .sup.15 N-urea is useful in the detection of Helicobacter pylori (Jicong et al. .sup.15 NH.sub.4.sup.+ excretion test: a new method for detection of Helicobacter pylori infection. J. Clin. Microbiol. 1992;30:181-84). Hamilton has described a method for detection of ammonia in the breath after ingestion of unlabeled urea (U.S. Pat. No. 4,947,861). This method is based on the premise that a substantial fraction of absorbed ammonia escapes metabolism by the liver (to urea) and therefore can be measured in the breath. Quite apart from the fact that this premise is not well supported by our current understanding of normal physiology, this method has the drawback of requiring a cumbersome apparatus for collection and analysis of breath ammonia.
All prior art methods focused on what until now was the only previously known target for urease reaction, mainly urea and its breakdown products i.e. CO.sub.2 (detection in the breath) and ammonia (detection in the breath or urine) In contrast, the present inventors have discovered other hitherto unknown substrates for the enzyme urease which yield derivatives that can be detected in the blood or urine and serve as unique diagnostic markers for the infection.